Contents

Introduction

Goal: to measure the proportion of a protein in the soluble versus insoluble state. Typical assays seem to use antibody probes against the supernatant and pellet of a standard lysis.

Principle

The basic method of all assays I've seen is to lyse cells into an aqueous buffer, spin down the pellet, pull off the supernatant and store it as the soluble fraction, then solubilize proteins remaining in the pellet using a solubilization buffer containing various detergents and denaturing agents (e.g. SDS, urea), spin down the pellet again, and pull off the supernatant and store it as the insoluble fraction.

Questions:

How do you ensure that you've preserved the composition of total protein in each fraction?

Extract in the same amount of buffer in each case, and load identical amounts of each fraction.

Control: Do the lysis in solubilization buffer, and save that fraction as total protein. Compare total protein to soluble + insoluble protein.